Catalogue # Aliquot Size

E12-11G -05 5 µg E12-11G -10 10 µg E12-11G -20 20 µg

EIF2AK4 (), Active Recombinant human protein expressed in Sf9 cells

Catalog # E12-11G

Lot # E 156 -1

Product Description Specific Activity

Recombinant human EIF2AK4 (192-1024) was expressed by baculovirus in Sf9 insect cells using an N-terminal GST 180,000 tag. The accession number is NM_001013703 . 135,000 Gene Aliases 90,000 GCN2; KIAA1338 45,000 Formulation (cpm) Activity 0

Recombinant protein stored in 50mM Tris-HCl, pH 7.5, 0 125 250 375 500 150mM NaCl, 10mM glutathione, 0.1mM EDTA, 0.25mM Protein (ng)

DTT, 0.1mM PMSF, 25% glycerol. The specific activity of EIF2AK4 was determined to be 14 nmol/min/mg as per activity assay protocol. Storage and Stability Purity Store product at –70 oC. For optimal storage, aliquot target into smaller quantities after centrifugation and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles. The purity of EIF2AK4 was determined to be >95% by Scientific Background densitometry, approx. MW 132kDa . EIF2AK4 or eukaryotic translation initiation factor 2 alpha 4 belongs to a family of that phosphorylate the alpha subunit of eukaryotic translation initiation factor-2 to downregulate protein synthesis in response to varied cellular stresses (1). The EIF2AK4 eIF2-alpha kinase regulates fatty-acid homeostasis in the liver during deprivation of an essential amino acid (2). EIF2AK4 (GCN2), Active Recombinant protein expressed in Sf9 cells References

1. Berlanga, J. J. et.al: Characterization of a mammalian Catalog Number E12-11G homolog of the GCN2 eukaryotic initiation factor 2-alpha Specific Activity 14 nmol/min/mg kinase. Europ. J. Biochem. 265: 754-762, 1999. Specific Lot Number E156-1 2. Guo, F. et.al: The GCN2 eIF2-alpha kinase regulates fatty- Purity >95% acid homeostasis in the liver during deprivation of an Concentration 0.1 µ g/ µl essential amino acid. Cell Metab. 5: 103-114, 2007. Stability 1yr At –70 oC from date of shipment o Storage & Shipping Store product at –70 C. For optimal storage, aliquot target into smaller

quantities after centrifugation and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles. Product shipped on dry ice.

To place your order, please contact us by phone 1-(604)-232-4600, fax 1-604-232-4601 or by email: [email protected] www.signalchem.com

FOR IN VITRO RESEARCH PURPOSES ONLY. NOT INTENDED FOR USE IN HUMAN OR ANIMALS. Activity Assay Protocol

Reaction Components

Active Kinase (Catalog #: E12-11G ) [33 P]-ATP Assay Cocktail Active EIF2AK4 (0.1 µg/ µl) diluted with Kinase Dilution Prepare 250 µM [33 P]-ATP Assay Cocktail in a designated Buffer IV (Catalog #: K24-09 ) and assayed as outlined radioactive working area by adding the following in sample activity plot. (Note: these are suggested components: 150 µl of 10mM ATP Stock Solution (Catalog #: working dilutions and it is recommended that the A50-09 ), 100 µl [ 33P]-ATP (1mCi/100 µl), 5.75ml of Kinase Assay researcher perform a serial dilution of Active EIF2AK4 for Buffer II (Catalog #: K02-09 ). Store 1ml aliquots at –20 oC. optimal results). Kinase Dilution Buffer IV (Catalog #: K2 4-09 ) 10mM ATP Stock Solution (Catalog #: A50-09 ) Kinase Assay Buffer II (Catalog #: K02-09 ) diluted at a Prepare ATP stock solution by dissolving 55mg of ATP in 10ml 1:4 ratio (5X dilution) with 50ng/ µl BSA solution. of Kinase Assay Buffer II (Catalog #: K02-09 ). Store 200 µl aliquots at –20 oC. Kinase Assay Buffe r II (Catalog #: K02 -09 ) Substrate (Catalog #: R55-58 ) Buffer components: 25mM MOPS, pH 7. 2, 12.5mM β- RS Repeat Peptide (GRSRSRSRSRSRSRSR) diluted in distilled glycerol-phosphate, 20mM MgC1 2, 12.5mM MnC1 2, H2O to a final concentration of 1mg/ml. 5mM EGTA, 2mM EDTA. Add 0.25mM DTT to Kinase Assay Buffer prior to use.

Assay Protocol

Step 1. Thaw [33P]-ATP Assay Cocktail in shielded container in a designated radioactive working area. Step 2. Thaw the Active EIF2AK4, Kinase Assay Buffer, Substrate and Kinase Dilution Buffer on ice. Step 3. In a pre-cooled microfuge tube, add the following reaction components bringing the initial reaction volume up to 20 µl: Component 1. 10 µl of diluted Active EIF2AK4 (Catalog #E12-11G) Component 2. 5µl of 1mg/ml stock solution of substrate (Catalog #R55-58)

Component 3. 5µl of distilled H2O

Step 4. Set up the blank control as outlined in step 3, excluding the addition of the substrate. Replace the substrate with an equal volume of distilled H 2O. Step 5. Initiate the reaction by the addition of 5 µl [ 33P]-ATP Assay Cocktail bringing the final volume up to 25 µl and incubate the mixture in a water bath at 30 oC for 15 minutes. Step 6. After the 15 minute incubation period, terminate the reaction by spotting 20 µl of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper. Step 7. Air dry the pre-cut P81 strip and sequentially wash in a 1% phosphoric acid solution (dilute 10ml of phosphoric acid and make a 1L solution with distilled H2O) with constant gentle stirring. It is recommended that the strips be washed a total of 3 intervals for approximately 10 minutes each. Step 8. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter. Step 9. Determine the corrected cpm by removing the blank control value (see Step 4) for each sample and calculate the kinase specific activity as outlined below.

Calculation of [P 33]-ATP Specific Activity (SA) (cpm/pmol)

Specific activity (SA) = cpm for 5 µl [33P] -ATP / pmoles of ATP (in 5 µl of a 250 µM ATP stock solution, i.e., 1250 pmoles)

Kinase Specific Activity (SA) (pmol/min/ µµµg or nmol/min/mg)

Corrected cpm from reaction / [(SA of 33P-ATP in cpm/pmol)*(Reaction time in min)*( amount in µg or mg)]*[(Reaction Volume) / (Spot Volume)]

To place your order, please contact us by phone 1-(604)-232-4600, fax 1-604-232-4601 or by email: [email protected] www.signalchem.com

FOR IN VITRO RESEARCH PURPOSES ONLY. NOT INTENDED FOR USE IN HUMAN OR ANIMALS.